The present invention is generally in the area of methods and systems for treatment of disorders such as cancer with biologically active agents produced naturally by cells in extremely small quantities, using genetically engineered host cells or natural cells that secrete a substance naturally implanted in biodegradable polymeric matrices.
One of the difficulties in treatment of conditions such as cancer using protein or other biological modifiers is the need for large quantities of the therapeutic agent to be delivered over an extended period of time. For most of the compounds discovered during research on complex pathways or unique tissues, it has not been possible, or has not been commercially feasible, to produce the compounds in sufficient quantity to treat the disorders. Numerous examples of these compounds, especially proteins, have been reported. One prominant example is angiostatin, a naturally occurring anti-angiogenic peptide identified by researchers at Children""s Medical Center in Boston, Mass. Although extremely promising in mice (O""Reilly et al., Science 285(5435):1926-8 1999), the inability of the developers to produce large quantities of the peptide has proven to be a major stumbling block to conducting clinical trials for treatment of cancer.
Mullerian Inhibiting Substance (MIS) is another biological with great potential for treatment of cancer. MIS is produced by the fetal testis and causes the regression in males of the Mxc3xcillerian duct, the forerunner of the female reproductive ducts. MIS has been shown to have great potential as a treatment for ovarian carcinomas (Chin et al, Cancer Research. 51:2101-2106, 1991; Masiakos, et al, Clinical Cancer Research, 5(11):3488-99 1999) which are derived from embryonic Mxc3xcllerian structures. Recombinant human MIS (rhMIS) produced in Chinese hamster ovary cells (CHO) in multiple roller bottles has antiproliferative activity against several human carcinoma cell lines (Chin, et al, 1991). Recently, it was also reported that rhMIS specifically binds to a functional heteromeric serine threonine (Teixeira, et al., Androl. 17(4):336-41 1996; Teixeira et al., Endocrinology Jan;137(1):160-5 1996) receptor on the surface of human ovarian cancer ascites cells and inhibits the growth in vitro of these cells and of cells obtained directly from women with Stage III and IV disease (Masiakos et al., 1999). See also, U.S. Pat. Nos. 4,404,199, 4,487,833, 4,510,131, 4,753,794, 4,792,601, 5,011,687, 5,198,420 and 5,661,126 to Donahoe, et al., the teachings of which are incorporated by reference herein.
The Pediatric Surgical Research Laboratories has tested the hypothesis that MIS will be a useful therapeutic agent for certain epithelial ovarian cancers in a number of in vitro studies described below, but only limited trials have been conducted in vivo. A major obstacle has been purifying sufficient recombinant protein of suitable potency and homogeneity for patient use.
Late stage epithelial ovarian cancer is a common and highly lethal gynecologic malignancy. Despite advances in treatment over the past two decades substantial improvement in overall survival has been slow and incremental and a high mortality remains. The coelomic epithelium, which invaginates to form the Mxc3xcllerian duct, is also the origin of these highly lethal human ovarian cancers. The hypothesis that MIS is a therapeutic for these Mxc3xcllerian derived tumors is predicated on previous observations in which partially purified bovine MIS (Donahoe et al., J Surg Res. 23: 141-8, 1977) suppressed growth of a single human ovarian cancer cell line in monolayer culture (Donahoe et al., Science. 205:913-5, 1979), in stem cell assays (Fuller et al., J Clin Endocr Metab. 54:1051-5, 1982), and in vivo in nude mice (Donahoe et al., Ann Surgery. 194:472-80,1981). Additionally, bovine MIS inhibited the growth of a large number of primary ovarian, Fallopian, and uterine carcinomas obtained directly from patients and tested in colony inhibition assays in soft agar (Fuller et al., Gynecol. Oncol. 22:135-148, 1985). After purifying bovine MIS (Budzik et. al., Cell 34: 307-314, 1983), the bovine and human MIS cDNAs and genomic human MIS (Cate et al., Cell. 45: 685-98 1986) were cloned. The human gene was used to produce highly purified recombinant human MIS (rhMIS) (Cate et. al., Cold Spring Harbor Symp Quant Biol 51 Pt 1:641-7 1986; MacLaughlin et al., Methods Enzymol. 198: 358-69, 1991) to which monoclonal and polyclonal antibodies were raised for use in a sensitive ELISA (Hudson et al., J Clin Endocrinol Metab. 70: 16-22, 1990; Lee et al J Clin Endocrinol Metab. 81:571-69, 1996). The rhMIS, which is now produced in a series of roller bottles and purified from the media (Ragin et al, Protein Expression and Purification, 1992; 3(3):236-45), was shown to inhibit three human carcinoma cell lines of Mxc3xcllerian origin (Chin et al., 1991), as well as a human ocular melanoma cell line (Parry et al., Cancer Res. 52:1182-6, 1992), in vitro and in vivo, in a dose dependent manner (Chin et al., 1991; Boveri et al., Int J Oncology. 2; 135-44, 1993). In order to scale up production beyond the roller bottle capacity which suites academic needs, a clonal line of MIS-producing transfected CHO cells (CHO, B9) was transferred to CHO B9 seeded bioreactors, for scale up to complete the phase I trials. Purification protocols for the bioreactor produced protein have been designed (Ragin et al, 1992), but modifications to improve purification protocols to enhance recovery and cioactivity have not resulted in production of sufficient quantities.
It is important to note that like the other members of the TGFxcex2 family, the bioactive purified protein is not a single polypeptide chain but a proteolytically cleaved molecule. MIS is primarily processed at residue 427, producing 110 kDa amino-terminal and 25 kDa carboxyterminal disulfide bond reduction sensitive homodimers (Pepinsky et al, J. Biol. Chem. 1988; 263:18961-4.; MacLaughlin et al, Endocrinology Jul;131(1):291-6 1992). Although the carboxy terminus is the active domain of rhMIS in vitro, it has not been shown to be active in vivo. The non-covalent association of the amino and carboxy termini is presumed to prevent the rapid clearance characteristic of the C terminus in vivo. Therefore, MIS to be administered to patients is being produced as a cleaved but non-dissociated complex. Unfortunately, the immunoaffinity purification protocols result in aggregation, given the hydrophobic character of MIS, with a product of reduced potency and low yield, making a process consistent with general manufacturing practices problematic. Moreover, alternative systems which are more efficient for large scale in vitro production, such as bacterial, yeast, or insect cell expression systems, have not been successful for the production of biologically active preparations of MIS.
It is therefore an object of the present invention to provide methods and reagents for production of clinically effective amounts of therapeutic biologicals, especially proteins such as MIS, in vivo.
It is a further object of the present invention to provide methods and reagents for production of biologics in vivo, where the production can be discontinued if appropriate.
It is a still further object of the present invention to provide methods and reagents for treatment of a variety of disorders characterized by the proliferation of abnormal tissue, including malignant and benign neoplasias, vascular malformations, inflammatory conditions including restenosis, infection, keloid formation and adhesions, congenital or endocrine abnormalities and other conditions that produce abnormal growth.
Normal cells, such as fibroblasts or other tissue or organ cell types, are genetically engineered to express biologically active, therapeutic agents, such as proteins that are normally produced in small amounts, for example, MIS, Herceptin(trademark), interferons, and Endostatin(trademark), or naturally produced compounds. These cells are seeded into a matrix for implantation into the patient to be treated. Cells may also be engineered to include a lethal gene, so that implanted cells can be destroyed once treatment is completed. Cells can be implanted in a variety of different matrices. In a preferred embodiment, these matrices are implantable and biodegradable over a period of time equal to or less than the expected period of treatment, during which the engrafted cells form a functional tissue producing the desired biologically active agent for longer periods of time. Representative cell types include tissue specific cells, progenitor cells, and stem cells. Matrices can be formed of synthetic or natural materials, by chemical coupling at the time of implantation, using standard techniques for formation of fibrous matrices from polymeric fibers, and using micromachining or microfabrication techniques.
These devices and strategies are used as delivery systems, which may be implanted by standard or minimally invasive implantation techniques, for any number of parenterally deliverable recombinant proteins, particularly those that are difficult to produce in large amounts and/or active forms using conventional methods of purification, for the treatment of a variety of conditions that produce abnormal growth, including treatment of malignant and benign neoplasias, vascular malformations (hemangiomas), inflammatory conditions, keloid formation and adhesion, endometriosis, congenital or endocrine abnormalities, and other conditions that can produce abnormal growth such as infection. Efficacy of treatment with the therapeutic biologicals is detected by determining specific criteria, for example, cessation of cell proliferation, regression of abnormal tissue, or cell death.
The examples demonstrate the use of this method with a tissue specific biological modifier, MIS. Genetically engineered CHO cells were grown on implantable polymeric meshes and the levels of secreted rMIS measured. The polymeric meshes with CHO cells seeded therein were then implanted in vivo and serum levels of rMIS measured. Data show very high levels of rMIS over prolonged time periods. These animals were then implanted with human ovarian cell lines and tumor regression measured in the presence of the MIS-producing cells. The implanted cells significantly inhibited the tumor cell proliferation.